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479

Asian-Aust. J. Anim. Sci.


Vol. 24, No. 4 : 479 - 484
April 2011
www.ajas.info

Oxidative Stress and Antioxidant Status during


Transition Period in Dairy Cows
N. Sharma, N. K. Singh*, O. P. Singh, V. Pandey and P. K. Verma
Division of Veterinary Clinical Medicine & Jurisprudence, F.V.Sc, SKUAST-J, India
ABSTRACT : The study was conducted on 20 Holstein X Sahiwal cross bred dairy cows, with an average milk production of
2,752113.79 liters in 2845.75 days during a single lactation, that were divided in to two groups of 10 animals. We investigated the
oxidative stress and antioxidant status during the transition period in dairy cows. In this study, plasma level of MDA was considered as
an indicator of lipid peroxidation and SOD, catalase, GSH and GSHPx as antioxidants. The lipid peroxidation was significantly
(p<0.001) higher in cows during early lactation as compared to the cows in advanced pregnancy. A significant positive correlation (r =
+0.831, p<0.01) was determined between MDA and catalase in early lactating cows. In early lactating cows, blood glutathione was
significantly lower than in advanced pregnant cows. However, early lactating cows showed non-significant negative correlation for all
antioxidant enzymes with lipid peroxidation. In conclusion, dairy cows seemed to have more oxidative stress and low antioxidant
defense during early lactation or just after parturition than advanced pregnant cows, and this appears to be the reason for their increased
susceptibility to production diseases (e.g. mastitis, metritis, retention of fetal membranes etc.) and other health problems. (Key Words :
Antioxidative Enzymes, Oxidative Stress, Production Diseases, Transition Period, Dairy Cow)

INTRODUCTION
The transition period between late pregnancy and early
lactation (also called the periparturient period) certainly is
the most interesting stage of the lactation cycle. Although
the length of time classified as the transition period has
been defined earlier by different authors, but we defined
this as last 4 weeks before parturition to first 4 weeks after
parturition. As stated by Goff and Horst (1997), The
transition from the pregnant, non-lactating state to the nonpregnant, lactating state is too often a disastrous experience
for the cows. Nutrition and management of cows during the
transition period has received tremendous interest lately.
Periparturient period is especially critical for health and
subsequent performance of dairy cows (Shanks et al., 1981).
Dairy cattle are more susceptible to a variety of metabolic
and infectious diseases during the transition period
compared with peak lactation (Sordillo et al., 2007).
Host defense mechanisms can be compromised directly
* Corresponding Author : N. K Singh. Lab of Stem Cells &
Regenerative Biotechnology, Dept. of Animal Biotechnology,
College of Animal Life Sciences, Kangwon National University,
Chuncheon, 200-701, Korea. Tel: +82-33-250-8637, Fax: +82-33251-7719, E-mail: singh@kangwon.ac.kr
Received June 15, 2010; Accepted November 25, 2010

because of numerous physiological and environmental


factors during the transition period. For example,
physiological stresses associated with rapid differentiation
of secretory parenchyma, intense mammary gland growth,
and the onset of copious milk synthesis and secretion are
accompanied by a high energy demand and an increased
oxygen requirement (Gitto et al., 2002). This increased
oxygen demand augments the production of oxygen-derived
reactants, collectively termed reactive oxygen species
(ROS). Although, oxygen is essential for all aerobic
organisms, has been termed the oxygen paradox.
Excessive production of free radicals and concomitant
damage at cellular and tissue levels are controlled by
cellular antioxidants defense systems. Antioxidants can be
broadly defined as any substance that delays, prevents or
removes oxidative damage to a target molecules (Halliwell
and Gutteridege, 2007). The preventive body antioxidative
defense systems can be accomplished by enzymatic (e.g.
SOD, GSHPx and Catalase) and non-enzymatic
mechanisms (e.g. Vitamin E and Selenium). Excessive
production of free radicals and ROS, and/or a decrease in
body antioxidant defense, lead to damage of biological
macromolecules and disruption of normal metabolism and
physiology (Trevisan et al., 2001). When ROS are produced
faster than they can be safely neutralized by antioxidant

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Sharma et al. (2011) Asian-Aust. J. Anim. Sci. 24(4):479-484

mechanisms, oxidative stress results. Therefore, an


imbalance between increased production of ROS and
reduced availability of antioxidant defenses near the time of
parturition increases oxidative stress and may contribute to
periparturient disorders in dairy cows (Waller, 2000; Gitto
et al., 2002). Unfortunately, oxidative stress as it is not a
classical disease, does not exhibit a specific clinical picture.
It has been observed that during the transition period cows
can experience oxidative stress (Formigoni et al., 1997;
Ronchi et al., 2000), which may contribute to periparturient
disorders and may be associated with metabolic diseases
(Ronchi et al., 2000). The determination of products of
peroxidative damage to macromolecules, and antioxidant
substances like glutathione and enzymes (e.g. SOD, GSHPx
and Catalase) are useful markers for the oxidative stress and
antioxidant status respectively. The oxidative destruction of
lipids (lipid peroxidation) is a destructive, self perpetuating
chain reaction, releasing malonyl aldehyde (MDA) as the
end product. However, as per our knowledge, literature on
the oxidative stress and antioxidant status during transition
period though replete but in Indian perspective there are
meager reports has been figured out so far.
The present study was planned with the view of the
possible oxidative stress involved during transition period;
it was decided to estimate the levels of MDA and SOD,
GSHPx, catalase and GSH as an index of oxidative stress
and antioxidative status respectively, and to see whether any
difference existed in these parameters during advanced
pregnancy and early lactation.

blood samples were collected from all cows in the morning


on the same day.

Collection of blood samples


Five ml blood sample were taken from jugular vein
using heparinized capped 5 ml vials and immediately placed
in an ice bath, where they were stored until they were
processed, within approximately 2 h from withdrawal. The

Oxidative stress
Changes in the oxidative stress and antioxidant status in
both the groups has been presented (Tables 1 and 2). The
lipid peroxidation (plasma MDA concentration) was
significantly (p<0.001) higher in early lactating cows

Biochemical analysis
All analyses were done with in 2 h after sample
collection. Extend of lipid peroxidation was estimated in
33% of erythrocyte lysate as the concentration of
thiobarbituric acid reactive product malondialdehyde
(MDA) by the method of Ohkawa et al. (1979). The values
of lipid peroxidation were expressed as (nm) nano moles of
MDA produced/g Hb/h using a molar extinction coefficient
of pure MDA as 1.56105. The plasma catalase (CAT)
activity was measured as per method described by the Aebi
(1983). In brief 20 l of 1% erythrocyte lysate was
incubated in 1.0 ml of 30 mM H2O2 at 37C and decrease in
absorbance was noted every 10 sec interval for one min. at
240 nm in a UV spectrophotometer (Schimadzu UV-1208
UV-VIS, Japan). The catalase activity was expressed as
moles of H2O2 decomposed/min/mg Hb using 36 as molar
extinction coefficient of H2O2. The activity of superoxide
dismutase (SOD) in 1% erythrocyte lysate was determined
by the method of Marklund and Marklund (1974). The
assay is based on the ability of SOD to exhibit the
autooxidation of pyrogallol in presence of EDTA. The
values were expressed as units/mg Hb. The blood
glutathione
(GSH)
levels
were
measured
spectrophotometrically by using method described by the
Beutler et al. (1963) and hemoglobin (Hb) by
Cyanomethoemoglobin method as described by Gowenlock
(1996). Changes in the optical density after reaction with
MATERIAL AND METHODS
DTNB reagent was estimated and final values were
extrapolated on the standard curve of glutathione and GSH
Study design and animals
concentration expressed as g/ml. The plasma glutathione
The study was conducted in the month of December, peroxidase (GSH-Px) activity was determined according to
2008, at organized dairy farms of northern and central India. the method of Hefeman et al. (1974). The rate of oxidation
Twenty Holstein X Sahiwal cross bred dairy cows aged of GSH by H2O2 was used as measure of GSHPx activity
from 26 months to 7 years and in 1 to 5 parity with an and expressed as units/mg Hb.
average milk production 2,752113.79 liters in 2845.75
days of corresponding lactation, were used in the study. Statistical analysis
Twenty animals were divided randomly into two groups A
All datas were analysed by using Students t-test to
and B, each group contain 10 animals. Group A cows were know the significance values between the groups. Pearsons
in last 4 weeks of pregnancy i.e. advanced pregnancy stage, correlations between level of peroxidation and activity of
while group B cows were in first 4 weeks of lactation i.e. antioxidative enzymes were calculated and, where
early lactation stage. All the cows were tie stall feeder and significant, are reported in the text. All statistical parameters
maintained on concrete floor. All the cows were examined were calculated as per the Snedecor and Cochran (1994).
for mastitis before collection of blood samples and selected
only mastitis free cows in the study.
RESULTS

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Sharma et al. (2011) Asian-Aust. J. Anim. Sci. 24(4):479-484

Table 1. Oxidative stress and antioxidant status in advanced pregnant (group A) and early lactating cows (group B) (Values are
expressed as meanSE)
Group

No. of animals

MDA

Cat

SOD

GSH

GSHPx

Hb

10

7.590.72

42.526.98

6.990.45

71.8111.41

1.930.22

11.620.50

10

13.342.68**

48.336.55

6.370.72

28.996.58*

1.190.13

12.080.32

* Denotes significant difference at p<0.002. ** Denotes significant difference at p<0.001.

Table 2. Correlation between oxidative stress and antioxidative status in advanced pregnant (group A) and early lactating cows
(group B)
Group
A
B

No. of Animals

MDA

10
10

1.00
1.00

CAT

SOD

0.831**
-0.553

GSH

-0.155
-0.231

0.336
-0.196

GSHPx
-0.330
0.350

** Correlation is significant at the 0.01 level (2-tailed).

(group B) as compared to advanced pregnant cows (group


A). The meanSE plasma level of MDA in advanced
pregnant dairy cows was 7.590.72 nm of MDA produced/g
Hb/h, which was nearly double in early lactating cows,
13.342.68 nm of MDA produced/g Hb/h. A significant
positive correlation (r = +0.831, p<0.01) was determined
between MDA and catalase in group A cows (Figure 1),
while negative correlation (r = -0.553) was in group B
animals between oxidative peroxidation (MDA) and
antioxidative status (CAT).

A significant positive correlation was detected between


MDA production and CAT activity in the animals of group
A (Figure 1). The activity of blood GSH was also increased
with the increased peroxidation (Table 2), while SOD and
GSHPx activity had negative correlation with MDA
production of group A cows. In early lactating cows all
antioxidant enzymes had non-significant negative
correlation with lipid peroxidation (Table 2).

Anti-oxidative status
In early lactating cows, meanSE of blood glutathione
(GSH) was 28.996.58 g/ml, which was significantly
lower than that of advanced pregnant cows, 71.8111.41
(Table 1). The meanSE of catalase activity was nonsignificantly higher in early lactating cows. The activities of
other anti-oxidative markers like SOD and GSHPx were
nearly similar in both the groups during transition period.

The role of antioxidants in health and disease was


studied extensively in both human and animal medicine
(Valko et al., 2007). Dairy cows undergo massive metabolic
adaptations during the onset of lactation, and it was
postulated that some of these physiological events may
negatively impact the health of the dairy cows (Sordillo et
al., 2009). Lipid peroxidation is one of important
consequences of oxidative stress (Kumaraguruparan et al.,
2002). The determination of lipid peroxidation products

DISCUSSION

Cat
140
120

MDA

100
80
60
40
20
0
0

10

12

CAT

Figure 1. Showing the positive correlation between MDA and CAT during advanced pregnancy.

14

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Sharma et al. (2011) Asian-Aust. J. Anim. Sci. 24(4):479-484

allows for the estimation of the intensity of this process;


moreover, it can be used for the evaluation of oxidative
stress severity (Halliwell and Whiteman, 2004). Lipids are
the most susceptible for peroxidative damage due to low
energy necessary for the initiation of the process as well as
the presence of unsaturated bonds (Balasinska, 2004).
In the present study lipid peroxidation (plasma MDA
production) was significantly (p<0.001) higher in early
lactating cows than advanced pregnant cows, which was
nearly double. The findings of our study are in
corroboration with the reports of Saleh et al. (2007); they
used thiobarbituric acid reactive substances (TBARS)
values as a marker of lipid peroxidation in cattle. Oxidative
stress in cows is a contributory factor to increase disease
susceptibility (Sordill, 2005), since metabolic demands
associated with late pregnancy, parturition and initiation of
lactation would be expected to increase the production of
reactive oxygen species (ROS), resulting oxidative stress. A
relationship between oxidative stress (lipid peroxidation)
and antioxidant status (catalase) was found significantly
positive in advanced pregnant cows, while non-significant
negative correlation was found in early lactating cows.
Saleh et al. (2007) have also been reported that depletion of
antioxidant activity and increase oxidative stress during
periparturient period that simulates our results. Stress due to
calving has a greater effect on this imbalance in addition to
decreased feed intake and plasma level of important
nutritional antioxidants/co-factors due to increased demand
of minerals and vitamins during advanced pregnancy and
more drainage in the colostrum. Unfortunately, it has not
been well known that how oxidative stress can affect health
and well-being, particularly during the times of high
metabolic activity. The performance of high yeilding dairy
cattles can be optimized to a certain extent by
supplementing diets with optimal levels of micronutrients
with antioxidant capabilities. However, oxidative stress
continues to be a problem in transition cows. Innovative
approaches to combat the progression of stress and to
enhance the antioxidant defense mechanisms of dairy cattle
during times of increased metabolic demands seems to be
pertinent (Sordillo and Aitken, 2009).
In the present findings, blood GSH was significantly
lower in early lactating cows than that of advanced pregnant
cows. In contrast, catalase activity was not significantly
higher in early lactating cows. However, Aitken et al.
(2009) had reported that activities of glutathione peroxidase
(GSHPx1 and GSHPx4) were increased during early
lactation. The activities of other anti-oxidative markers like
SOD and GSHPx were nearly similar in both the groups
during transition period during the study. Holbrook and
Hicks (1978) also observed no significant differences in the
SOD concentration throughout the lactation of nonmastitic
cows.

The activity of blood GSH was increased with the


increased lipid peroxidation, while SOD and GSHPx
activity had negative correlation with MDA production in
group A cows. Therefore, an imbalance between increased
production of ROS and reduced availability of antioxidant
defense near the time of parturition might increase oxidative
stress and may contribute to periparturient disorders in
dairy cows (Waller, 2000; Gitto et al., 2002). Increased
incidence of disease during the periparturient period is
related directly to numerous genetic, physiological, and
environmental factors that can compromise the cows
immunological defenses (Sordillo, 2005). A relationship
between the physiological changes associated with
parturition and a loss in overall antioxidant potential was
established in both humans and dairy cows (Bernabucci et
al., 2005; Sordillo et al., 2007). The possibility that
oxidative stress during the transition period particularly
during parturition may be a major underlying cause of
inflammatory and immune dysfunction in dairy cattle has
earlier been supported by various studies conducted either
in vivo or in vitro (Sordillo and Aitken, 2009).
Superoxide dismutase, catalyzes the dismutation of
superoxide radicals into hydrogen peroxide and molecular
oxygen. Hydrogen peroxide degrades further to water by
other antioxidant enzymes, such as glutathione peroxidase
and catalase. In mammalian cells, there are three types of
superoxide dismutase: cytosolic CuZn superoxide dismutase
(CuZnSOD), mitochondrial manganesesuperoxide dismutase
(MnSOD), and extracellular superoxide dismutase
(ECSOD). Glutathione peroxidase proteins catalyze the
reduction of organic hydroperoxides, lipid peroxides, and
hydrogen peroxide, using glutathione as the reducing agent,
thereby also protecting cells from oxidative damage
resulting from normal oxidative metabolism. There are four
known GSHPx that contain selenocysteine at the active site.
In early lactating cows all antioxidant enzymes had nonsignificant negative correlation with lipid peroxidation.
During advanced pregnancy and early lactation increased
demand of micronutrients donot usually full fill the
resulting deficiencies occurring due to natural protective
substances or excess exposure to stimulators of reactive
oxygen metabolites (ROM), and this might results in high
lipid peroxidation and decreased level of antioxidant
enzymes. Significant correlation between antioxidant
supplementation and decreased incidence of mastitis (Weiss
et al., 1997; Allison and Laven, 2000) has been reported
previous, which supports our results and presumptions.
Decreased level of antioxidant enzymes during early
lactation in our study could be supported as oxidative stress
increases during parturition and early lactation stages.
Oxidative stress increase causes continuous increase in the
concentration of lipid peroxidation products and decrease in
the level of enzymatic and non-enzymatic antioxidants after

Sharma et al. (2011) Asian-Aust. J. Anim. Sci. 24(4):479-484


transiently increased activity to combat the toxic effects of
reactive oxygen species (ROS). The antioxidant enzymes
such as GSHPx and catalase might have important
functions in alleviating the toxic effects of ROS (Vannucchi
et al., 1997; Kale et al., 1999). Infection and tissue repair
are common even in well-managed dairy herds, and cows
may experience some degree of immune response,
especially after calving. Stress disease and induction of the
immune response increases nutrient requirements (Madsen
et al., 1990; Madsen, 1991) has been known. Inadequacies
of these nutrients required for both immunity and
antioxidant defense could impair function of both systems.
To optimize performance, oxidative stress in high producing
cows must be controlled by supplying all known
antioxidant nutrients and by minimizing effects of
substances that stimulate ROM. Significant correlation
between antioxidant supplementation and decreased
incidence of mastitis (Weiss et al., 1997; Allison and Laven,
2000) has already been reported. Conclusively, dairy cows
seemed to have more oxidative stress and low antioxidant
defense during early lactation or just after parturition than
advanced pregnant cows, and this seemed to be the probable
reason for their increased susceptibility to production
diseases (e.g. mastitis, metritis, retention of fetal
membranes etc.) and other health problems.
ACKNOWLEDGMENTS
First and Second authors has contributed equally and
the research jointly carried out partly at KNU, Chuncheon,
Korea and SKUAST-J, India.
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